1. Field of the Invention
The present invention is directed to a method for the separation and recovery of certain desired biological substances from liquids containing the same, and to devices for achieving such separation and recovery. More particularly, this invention is directed to the recovery in high concentration of such biological substances as desoxyribonucleic acid (DNA) from liquids containing the same, including broths, gels, or the like, employing an inventive centrifuge tube having affixed therein a porous selection means, as defined herein, to which the liquid is permeable, but which contains sites having an affinity for, and thus selectively binds, the DNA or other biological substance to the selection means. The bound substances may then be recovered from this porous material by elution.
2. Statement of Related Art
Recent advances in molecular biology and like disciplines have required faster and more accurate techniques in the recovery, purification and analysis of small amounts of biological substances such as DNA segments and proteins. Methods such as electrophoresis and other chromatographic separation processes have all required as a necessary step the separation and recovery of these biological materials from gels, broths, or like media, conventionally using either various types of adsorption columns such as ion exchange columns, recovery from gel slices by electroelution or freeze-squeeze techniques; or selective precipitation with e.g., acetone. More recently, an alternate method for recovering DNA from gels after electrophoretic separation has been provided which involves dissolving the desired gel slice in chaotropic salts such as sodium iodide (NaI), followed by selective binding of the DNA to glass beads, i.e. a suspension of silica matrix in water, which is then subjected to centrifugation. Proc. Natl. Acad. Sci. USA76 No. 2, pp. 615-619 (1979). In a similar fashion recent recovery methods include solubilization of the DNA-containing agarose gels, followed by addition of a "purification matrix" to the solution to adsorb the DNA alone. Removal of the adsorbed DNA by a wash buffer, and then centrifugation, recovers the DNA by this elaborate process. Bio-Radiatlons, No 73, Summer 1989 (Bio-Rad Laboratories, Richmond, Va.). See also "Molecular Cloning: A Laboratory Manual", Maniatis et al., pp. 466-467 (Cold Spring Harbor Laboratory, 1982), which also uses a centrifuge tube.
Additionally, various types of test tubes, pipettes, centrifuge tubes, concentrators and like devices in combination with integral filters, and use of these devices in the filtration separation of solids, particularly biological substances, from liquids, liquids from other liquids, and in certain chemical and biological test procedures are already described in the art. See, for example, U.S. Pat. No. 4,557,902 directed to a test tube containing various reagents, for detecting gases, vapors and aerosols, in combination with an internal filter disc separating the reagents. See, also Bio-Rad Bulletin 1402 (1987), (Bio-Rad Laboratories, Richmond, Va.) describing chromatographic spin-column devices and techniques for recovery of radiolabeled nucleotides as well as U S Pat. No. 3,732,981, which discloses columns with removable caps and "snap-off" tips. Acknowledged prior art devices in combination with a filter paper flush with the wall of the tube in a non-filter arrangement are also described, wherein the filter paper is impregnated with a combination of zinc powder, silica gel, inert quartz and silica gel impregnated with gold chloride. Also illustrative of a tube containing a filter medium placed along the axis of the tube rather than in a conventional filtering arrangement is the centrifuge tube of U.S. Pat. No. 4,600,507.
Typical of centrifuge tubes, pipettes and the like having porous membranes arranged across the tube to function as conventional filters are those shown in U.S. Pat. Nos. 4,483,825, to a centrifuge pipette, and 4,301,118 for a protein concentrator. U.S. Pat., Nos. 4,632,761 and 4,787,971 are cumulative thereto, as well as U.S. Pat. No. 4,832,851 directed to a highly complex multi-chamber device. See, also, Millipore.RTM. News, No. 12 (1989) (Millipore Corp., Bedford, Mass.), as well as corresponding product bulletins, disclosing "low binding membranes" for filtration through centrifuge tubes, which are described favorably as filters with minimum binding, thereby teaching away from membranes which would selectively bind desired biomolecules via centrifugation. Like devices include those shown by the Schleicher and Schuell publication "Sequences", Issue 30, Spring, 1989 (Schleicher and Schuell, Keene, NH, U.S.A.), disclosing their Centrex.RTM. filter device, and also the filtration tube of the Kontes catalog of Nov. 1, 1983, at page 219 (item K-413900). (Kontes Co., Vineland, N.J., U.S.A.)
In each device described above the membranes are employed solely as filtration devices, often incorporated in highly complex, intricately engineered devices, none of which teaches the concept of using in combination with a simple centrifuge tube arrangement, a porous selection means containing sites to selectively bind and thereby separate certain biological materials which can thereafter be eluted from the binding site in high concentration.
These methods and devices, while of varying effectiveness, are of limited commercial use in that they are characterized by such complexities as setting up columns; pipetting special reagents, as in the case of glass suspensions; processing only a few samples at a time, as for example, with gel slices; or various associated elution techniques which use toxic solvents, and/or provide low recovery of desired product. See, for example, "Molecular Cloning" (above), pp. 164-172, at p. 164, where it is noted that many methods have been developed to recover DNA from agarose gels, but that none is entirely satisfactory. With the growth of molecular biological research generally, and proposed genome projects in particular, increased facility in selectively separating and recovering a multiplicity of samples of DNA or other biomolecules which avoid such problems would thus be highly desirable.